The present invention relates to a protein having a transglutaminase activity, DNA which encodes for the protein, and a process for producing the protein. In particularly, the present invention relates to a process for producing a protein having a transglutaminase activity by a genetic engineering technique.
Transglutaminase is an enzyme which catalyzes the acyl transfer reaction of a .gamma.-carboxyamido group in a peptide chain of a protein. When such an enzyme react with the protein, a reaction of an .epsilon.-(.gamma.-Glu)-Lys forming reaction or substitution reaction of Gln with Glu by the deamidation of Glu can occur.
The transglutaminase is used for the production of gelled foods such as jellies, yogurts, cheeses, gelled cosmetics, etc. and also for improving the quality of meats [see Japanese Patent Publication for Opposition Purpose (hereinafter referred to as "J. P. KOKOKU") No. Hei 1-50382]. The transglutaminase is also used for the production of a material for microcapsules having a high thermal stability and a carrier for an immobilized enzyme. The transglutaminase is thus industrially very useful.
As for transglutaminases, those derived from animals and those derived from microorganisms (microbial transglutaminase; hereinafter referred to as "MTG") have been known hitherto.
The transglutaminases derived from animals are calcium ion-dependent enzymes which are distributed in organs, skins and bloods of animals. They are, for example, guinea pig liver transglutaminase [K. Ikura et al., Biochemistry 27, 2898 (1988)], human epidermis keratin cell transglutaminase [M. A. Philips et al., Proc. Natl. Acad. Sci. USA 87, 9333 (1990)] and human blood coagulation factor XIII (A. Ichinose et al., Biochemistry 25, 6900 (1990)].
As for the transglutaminases derived from microorganisms, those independent on calcium were obtained from microorganisms of the genus Streptoverticillium. They are, for example, Streptoverticillium griseocarneum IFO 12776, Streptoverticillium cinnamoneum sub sp. cinnamoneum IFO 12852 and Streptoverticillium mobaraense IFO 13819 [see Japanese Patent Unexamined Published Application (hereinafter referred to as "J. P. KOKAI") No. Sho 64-27471].
According to the peptide mapping and the results of the analysis of the gene structure, it was found that the primary structure of the transglutaminase produced by the microorganism is not homology with that derived from the animals at all (European Patent publication No. 0 481 504 Al).
Since the transglutaminases (MTG) derived from microorganisms are produced by the culture of the above-described microorganisms followed by the purification, they had problems in the supply amount, efficiency, and the like. It is also tried to produce them by a genetic engineering technique. This technique includes a process which is conducted by the secretion expression of a microorganism such as E. coli, yeast or the like (J. P. KOKAI No. Hei 5-199883), and a process wherein MTG is expressed as an inactive fusion protein inclusion body in E. coli, this inclusion body is solubilized with a protein denaturant, the denaturant is removed and then MTG is reactivated to obtain the active MTG (J. P. KOKAI No. Hei 6-30771).
However, these processes have problems when they are practiced on an industrial scale. Namely, when the secretion by the microorganisms such as E. coli and yeast is employed, the amount of the product is very small; and when MTG is obtained in the form of the inactive fusion protein inclusion body in E. coli, an expensive enzyme is necessitated for the cleavage.
It is known that when a foreign protein is secreted by the genetic engineering method, the amount thereof thus obtained is usually small. On the contrary, it is also known that when the foreign is protein is produced in the cell of E. coli, the product is in the form of inert protein inclusion body in many cases although the expressed amount is high. The protein inclusion body must be solubilized with a denaturant, the denaturating agent must be removed and then MTG must be reactivated.
It is already known that in the expression in E. coli, an N-terminal methionine residue in natural protein obtained after the translation of gene is efficiently cleaved with methionine aminopeptidase. However, the N-terminal methionine residue is not always cleaved in an exogenous protein.
Processes proposed hitherto for obtaining a protein free from N-terminal methionine residue include a chemical process wherein a protein having methionine residue at the N-terminal or a fusion protein having a peptide added thereto through methionine residue is produced and then the product is specifically decomposed at the position of methionine residue with cyanogen bromide; and an enzymatic process wherein a recognition sequence of a certain site-specific proteolytic enzyme is inserted between a suitable peptide and an intended peptide to obtain a fusion peptide, and the site-specific hydrolysis is conducted with the enzyme.
However, the former process cannot be employed when the protein sequence contains a methionine residue, and the intended protein might be denatured in the course of the reaction. The latter process cannot be employed when a sequence which is easily broken down is contained in the protein sequence because the yield of the intended protein is reduced. In addition, the use of such a proteolytic enzyme is unsuitable for the production of protein on an industrial scale from the viewpoint of the cost.
Conventional processes for producing MTG have many problems such as supply amount and cost. Namely, in the secretion expression by E. coli, yeast or the like, the expressed amount is disadvantageously very small. In the production of the fusion protein inclusion body in E. coli, it is necessary, for obtaining mature MTG, to cleave the fusion part with restriction protease after the expression. Further, it has been found that since MTG is independent on calcium, the expression of active MTG in the cell of a microorganism is fatal because this enzyme acts on the endoprotein.
Thus, for the utilization of MTG, produced by the gene recombination, on an industrial scale, it is demanded to increase the production of mature MTG free of the fusion part. The present invention has been completed for this purpose. The object of the present invention is to product MTG in a large amount in microorganisms such as E. coli.
When MTG is expressed with recombinant DNA of the present invention, methionine residue is added to the N-terminal of MTG. However, by the addition of the methionine residue to the N-terminal of MTG, there is some possibility wherein problems of the safety such as impartation of antigenicity to MTG occur. It is another problem to be solved by the present invention to produce MTG free of methionine residue corresponding to the initiation codon.